PROJECT SUMMARY/ ABSTRACT. Staphylococcus aureus and Pseudomonas aeruginosa are the two most common bacterial species associated with chronic lung infections in cystic fibrosis (CF). While historically S. aureus was commonly isolated in younger CF patients and then replaced by P. aeruginosa, it is now the bacterial pathogen most frequently detected in sputum samples from all CF patients. Consequently, there is a large time interval when both S. aureus and P. aeruginosa are found together in CF sputum samples. A number of studies including those from our group have shown that co-infection is associated with diminished lung function and pulmonary decline, however the molecular mechanism for this poor outcome is not known. While most reports have noted that P. aeruginosa readily kills S. aureus, we have found that the mucoid phenotype of P. aeruginosa aids in its co- existence with S. aureus. With respect to S. aureus and adaptation to the CF lung, other than the emergence of small colony variants and the mucoid phenotype, the factors intrinsic to S. aureus that promote chronic CF colonization?as well as those that engender co-existence with P. aeruginosa and elicit loss of CF respiratory function?are not well understood. We hypothesize that there are additional S. aureus genetic determinants that promote survival in the presence of P. aeruginosa in the CF lung environment. To begin to test this hypothesis, we have developed a new co-culture assay and tested 65 CF isolates of S. aureus (from 50 individuals with CF) obtained from the Emory CF Biospecimen Repository (CF-BR) and Boston Children's Hospital for their interactions with the non-mucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative and have categorized these isolates into 3 groups based on these interactions. These isolates have been subjected to whole genome sequencing (WGS) and we have the metadata associated with the CF patients at the time the samples were taken. Here we will take 3 complementary approaches to identify genetic factors of S. aureus that promote its interactions with P. aeruginosa. We will obtain and sequence ~200 additional longitudinal CF patient isolates of S. aureus banked in the Emory CF-BR. In particular, we will select isolates that differ in their P. aeruginosa co-infection status over several years of sample acquisition and identify SNP differences between genetically similar isolates. We will experimentally evolve S. aureus to survive in the presence of P. aeruginosa. WGS compared to the progenitor will identify genes responsible for resistance to P. aeruginosa. We will perform Tn-seq analysis of S. aureus on select clinical isolates in the presence of P. aeruginosa in co-culture. Mutants that are lost after interaction with P. aeruginosa will define genes that are critical for survival under these conditions. By the end of this study, we will have identified S. aureus genes that allow it to co-exist with P. aeruginosa; future characterization of these factors should increase our understanding of why co-infection correlates with worse patient outcome in CF.